Abstract Hox gene mutations in simpler organisms often resulting in dramatic homeotic transformations of one body part into another. They encode transcription factors that can initiate genetic cascades that drive the developmental destinies of segments. In mammals there are 39 Hox genes, in four clusters, divided into 13 functionally related paralog groups. It is necessary to mutate multiple Hox genes from multiple paralog groups to overcome redundancies and reveal previously hidden shared functions. To this end we have made mice with frameshift mutations in sets of adjacent Hox genes. By interbreeding we can dial down Hox function for multiple paralog groups while maintaining sufficient Hox11 expression to have a developing kidney to study. Mice with simultaneous frameshift mutation of twelve closely related Hox9,10,11 alleles show a very unexpected kidney phenotype. The cells of the mutant nephrons often show mixed identities, with co- expression of markers of more than one segment cell type. This was examined with an extensive battery of segment specific markers. Providing further confirmation, Hox mutation in Drosophila can also lead to de- repression of many genes normally expressed in other lineages, very similar to what we see in mice. In specific aim 1 we propose to further study the apparently confused, mixed identity character of the multi-Hox mutants using single cell RNA-seq and single cell ATAC-seq, to define the limits of the crossing of lineage boundaries and to search for underlying mechanisms. How many different cell type markers can be expressed by an individual cell? Do open/closed chromatin configurations in mutants reveal even more epigenetic plasticity than seen by RNA-seq? Are there perturbations in pathways that suggest mechanisms? In specific aim 2 we test the hypothesis that the observed mixed cell identity mutant phenotype is the result of disrupted Polycomb Repressive Complex (PRC) function. Cell type specific transcription factors initially establish repressed gene expression states, and then PRC complexes recognize and maintain their repression. Work in Drosophila has shown that Hox proteins and PRC proteins can co-bind to drive repression of inappropriate cell type genes. In this aim we carry out a series of Chip-seq experiments to compare wild type/mutant distributions of PRC1 (H2Aub1), PRC2 (H3K27me3), active (H3K4me3), Hoxa11 and Hoxd11 (using our epitope tagged mice), as well as Ezh2 and Ring1B PRC component proteins. In specific aim 3 we propose to test the hypothesis that Hox genes also function to reduce cell type plasticity in the adult. Hox genes generally continue to be expressed in the adult. The function for this has remained uncertain, although it has been proposed that it serves to maintain expression of the appropriate differentiation genes. We propose that it also serves to maintain repression of inappropriate differentiation genes. We propose to carry out single cell RNA-seq experiments on ischemia reperfusion wild type and Hox mutant injured kidneys, where cells normally de-differentiate and then re-differentiate, to test his hypothesis.